The conversion of human immunodeficiency virus (HIV) genomic RNA into DNA comprises multiple steps, each catalyzed by the multifunctional viral enzyme, reverse transcriptase (RT). Many inhibitors of HIV RT DNA polymerase activity have been discovered and ten of these are in current clinical use. In contrast, no inhibitors of HIV RT-associated RNase H are in clinical use; indeed, very few such inhibitors have even been identified. This application seeks support for an ongoing but currently unfunded collaboration between Michael Parniak (University of Pittsburgh School of Medicine) and Fluorous Technologies, Inc. (FTI). These collaborators bring complementary backgrounds and skills to this promising drug discovery project. Parniak is an expert in the biochemistry of HIV RT including the RNase H activity of this viral enzyme and has recently developed the first high throughput screen to identify inhibitors of RNase H. FTI is developing and commercializing innovative fluorous mixture synthesis technology initiated in the laboratories of consultant Dennis Curran (University of Pittsburgh, Department of Chemistry), and has demonstrated this technology by making libraries of the natural product mappicine. Screening of a preliminary mappicine library has identified a number of potent inhibitors of HIV RT RNase H. This application takes the first steps towards the long-range goal of discovering and developing mappicine-based antiretroviral agents that act by inhibition of RNase H. To this end, the specific aims of this Phase I STTR proposal are: (1) To screen an existing library of 560 mappicine analogs to identify inhibitors of HIV-1 RT-associated RNase H; (2) To design and synthesize on 10-20 mg scale a focused library of about 40-100 new analogs of the most active mappicine derivatives identified from Specific Aim 1; (3) To screen the focused mappicine analog library for inhibition of HIV-1 RT RNase H; and (4) To characterize the antiviral properties, viral resistance profiles and cytotoxicity of the most active compounds identified in Specific Aim 3. The proposed Phase I studies provide an aggressive "proof-of-concept" approach to validating mappicine RNase H inhibitors, and will provide a firm basis for subsequent Phase II preclinical development of this novel class of potential anti-retrovirals.